The dihydrochalcone phlorizin (phloretin 2′-glucoside, see FIG. 1) is the major phenolic glucoside found in apple trees. Phlorizin has a bitter taste that contributes to the characteristic flavour of cider (Whiting and Coggins 1975), and its dimerised oxidation products contribute to the colour of apple juices (Ridgway and Tucker 1997). However, since it was isolated from the bark of the apple tree in 1835 (Petersen 1835), phlorizin has attracted most scientific interest through its use as a pharmaceutical and tool for physiology research. Its principal pharmacological action is to produce renal glycosuria and block glucose transportation by inhibition of the sodium-linked glucose transporters (reviewed in Ehrenkranz et al. 2005). Phlorizin and its derivatives have also been shown to be an extremely effective antioxidants in vitro (Ridgway et al. 1996), and to have a range of bioactive functions such as inhibition of lipid peroxidation (Ridgway et al. 1997; Rezk et al. 2002), prevention of bone loss (Puel et al. 2005), enhancement of memory (Boccia et al. 1999), and inhibition of cancer cell growth (Veeriah et al. 2006).
Until recently phlorizin was believed to exist only in Malus species. However phloretin glycosides have been reported in the leaves of Australian native sarsaparilla (Smilax glyciphylla, Cox et al. 2005), sweet tea (Lithocarpus polystachyus, Dong et al. 2007) and at very low levels in strawberry fruit (Hilt et al. 2003). In apple trees, phlorizin is found primarily in the young shoots, roots, leaves and bark. In fruit, phlorizin is most abundant in the seeds, with intermediate levels in both the core and the skin, and the lowest level in the cortex. Variation has been assessed within apple trees, between orchards, between different cultivars and among mutants (Hunter and Hull 1993, Awad et al. 2000). Despite this information, little is known of the in planta function of phlorizin in apple tree physiology, although it has been suggested that it might act in apple tree growth and development (Zhang et al. 2007) or be an inhibitor of bacterial (MacDonald and Bishop 1952) or fungal growth (Gessler et al. 2006).
The molecular basis for production of phlorizin in planta has not been described. Phloretin is a product of the phenylpropanoid pathway (Watts et al. 2004), with conversion to its glucoside, phlorizin, likely to be catalysed by the action of a uridine diphosphate (UDP) glycosyltransferase (UGT). UGTs mediate the transfer of a sugar residue from an activated nucleotide sugar to acceptor molecules (aglycones). Plants contain large families of UGTs with over 100 genes being described in Arabidopsis. These genes have a common signature motif of 44 amino acids thought to be involved in binding of the UDP moiety of the activated sugar (Li et al. 2001). A phylogenetic analysis established the presence of distinct Groups (A-N) and Families (UGT71-92) of UGT genes in Arabidopsis (Ross et al. 2001) and this facilitated the characterisation of many new activities (Jackson et al. 2001; Lim et al. 2002; Jones et al. 2003; Messner et al. 2003; Lim et al. 2004; Lim et al. 2005). Although initially thought to be promiscuous enzymes, recent evidence suggests that their broad substrate specificity is limited by regio-specificity (Hansen et al. 2003; Lim et al. 2003), and in some cases UGTS have been shown to be highly specific (Fukuchi-Mizutani et al. 2003). Using a functional genomics approach we have identified and characterised a UGT from apple belonging to the previously uncharacterised UGT Family 88. We establish that MpUGT88A1 mediates the glycosylation of the dihydrochalcone phloretin to phlorizin which may indicate that other members of UGT Family 88 utilise similar substrates.
It would be beneficial to have a means to increase phlorizin levels in plants.
It is an object of the invention to provide improved compositions and methods for modulating activity and/or phlorizin content in plants or at least to provide the public with a useful choice.